Different drugs used to inhibit microbial growth act by inhibiting different targets. For example, the fluoroquinolone class of antibiotics act by inhibiting bacterial DNA synthesis. When used in treatment, fluoroquinolones are well absorbed orally, are found in respiratory secretions in higher concentrations than in serum and are concentrated inside macrophages. In addition, fluoroquinolones are well tolerated and have an excellent safety record in long-term therapy.
Antibiotic resistance, and in particular resistance to fluoroquinolones, has become a problem. Fluoroquinolone resistance in gram negative bacteria is principally caused by mutations affecting the target proteins of the drugs. In the case of fluoroquinolones, these targets are DNA gyrase and topoisomerase IV. In addition, mutations affecting regulatory genes such as marA, soxS or rob can cause fluoroquinolone resistance (Oethinger et al. 1998. J. Antimicrob. Chemother. 41: 111). Mar A is a transcriptional activator encoded by the marRAB operon involved in multiple antibiotic resistance (Alekshun et al. (1997) Antimicrob. Agents Chemother. 41, 2067-2075). The marRAB locus confers resistance to tetracycline, chloramphenicol, fluoroquinolones, nalidixic acid, rifampin, penicillin, as well as other compounds. However, marRAB does not encode a multidrug efflux system. Rather, it controls the expression of other loci important in directly mediating drug resistance, e.g., ompF, the gene for outer membrane porin, and the acrAB genes for the AcrAB efflux proteins.
AcrAB is a multidrug efflux pump (Nikaido, H. (1996) J. Bacteriol. 178, 5853-5859; Okusu et al. (1996) J. Bacteriol. 178, 306-308) whose normal physiological role is unknown, although it may assist in protection of cells against bile salts in the mammalian small intestine (Thanassi et al. (1997) J. Bacteriol. 179,2512-2518). The AcrAB operon is upregulated by MarA (Ma et al. (1995) Mol. Microbiol. 16, 45-55). Mutations in the repressor gene marR lead to overexpression of marA (Alekshun et al. (1997). Antimicrob. Agents Chemother. 41, 2067-2075; Cohen et al. (1993) J. Bacteriol. 175, 1484-492); Seoane et al. (1995) J. Bacteriol. 177, 3414-3419). The soxS gene encodes a MarA homolog (Alekshun et al. (1 997) Antimicrob. Agents Chemother. 41, 2067-2075; Li et al. (1996) Mol. Microbiol. 20, 937-945; Miller et al. (1996) Mol. Microbiol. 21, 441-448) which also positively regulates acrAB (Ma et al. (1996) Mol. Microbiol. 19, 101-112).
The AcrAB pump primarily controls resistance to large, lipophilic agents that have difficulty penetrating porin channels, such as erythromycin, fusidic acid, dyes, and detergents, while leaving microbes susceptible to small antibiotics that can diffuse through the channel, e.g., tetracycline, chloramphenicol, and fluoroquinolones (Nikaido. 1996. J. Bacteriology 178:5853). Recently, the AcrAB pump has been found to be important in mediating resistance to other drugs used to control microbial growth, e.g., non-antibiotic agents such as triclosan (FEMS Microbiol Left Sep. 15, 1998; 166: 305-9.
Microbes often become resistant to antibiotics and/or non-antibiotic agents. This can occur by the acquisition of genes encoding enzymes that inactivate the agents, modify the target of the agent, or result in active efflux of the agent. Enzymes that inactivate synthetic antibiotics such as quinolones, sulfonamides, and trimethoprim have not been found. In the case of these antibiotics and natural products for which inactivating or modifying enzymes have not emerged, resistance usually arises by target modifications (Spratt. 1994. Science 264:388). Improved methods for controlling drug resistance in microbes, in particular in microbes that are highly resistant to drugs, would be of tremendous benefit.
The present invention is based, at least in part, on the discovery that inactivation of the AcrAB locus makes even resistant microbial cells hypersusceptible to antibiotics and non-antibiotic drugs. Surprisingly, this is true even among highly resistant microbes which have chromosomal mutations that render them highly resistant to drugs.
Accordingly, in one aspect, the invention provides methods of treating an infection caused by a drug resistant microbe in a subject by administering a drug to which the microbe is resistant and an inhibitor of an AcrAB-like efflux pump to the subject such that the infection is treated.
In one embodiment, the drug is an antibiotic. In a preferred embodiment the antibiotic is selected from the group consisting of a fluoroquinolone and rifampin. In another embodiment, the drug is a non-antibiotic agent. In another embodiment, the drug is the non-antibiotic agent, triclosan.
In one embodiment, the inhibitor of an AcrAB-like efflux pump is administered prophylacticly. In another embodiment, the inhibitor of an AcrAB-like efflux pump is administered therapeutically.
In another aspect, the invention pertains to a method of treating a fluoroquinolone resistant infection in a subject comprising administering a fluoroquinolone and an inhibitor of an AcrAB-like efflux pump to the subject to thereby treat a fluoroquinolone resistant infection.
In another aspect, the invention pertains to a method of screening for compounds which reduce drug resistance comprising: contacting a microbe comprising an AcrAB-like efflux pump with a test compound and a indicator compound and measuring the effect of the test compound on efflux of the indicator compound to thereby identify compounds which reduce drug resistance by testing the ability of the test compound to inhibit the activity of an AcrAB efflux pump.
In one embodiment, the microbe is drug resistant. In a preferred embodiment, the microbial cell is highly drug resistant. In a more preferred embodiment, the microbe is highly resistant to fluoroquinolones. In another embodiment, the microbial cell comprises at least one mutation in a drug target gene. In another embodiment the microbial cell comprises at least two mutations in a drug target gene. In a preferred embodiment, a mutation is present in a gene selected from the group consisting of: gyrase (gyrA), topoisomerase (parC), RNA polymerase, and fabl.
In one embodiment, the subject assay includes detecting the ability of the compound to reduce fluoroquinolone resistance in a microbe.
In another aspect, the invention provides a method of screening for compounds which specifically inhibit the activity of an AcrAB-like efflux pump comprising:
i) contacting a microbe comprising an AcrAB-like efflux pump with a test compound and an indicator compound;
ii) testing the ability of the compound to inhibit the activity of an AcrAB-like efflux pump;
iii) testing the ability of the compound to inhibit the activity of a non-AcrAB efflux pump;
iv) and identifying compounds which inhibit the activity of an AcrAB-like efflux pump and non a non -AcrAB-like efflux pump to thereby identify compounds which specifically block an AcrAB-like efflux pump.
In yet another aspect, thand invention provides a method of enhancing the antimicrobial activity of a drug comprising: contacting a microbe that is highly resistant to one or more drugs with a drug to which the microbe is resistant and an inhibitor of an AcrAB-like efflux pump to thereby enhance the antimicrobial activity of a drug.
In one embodiment, the step of contacting occurs ex vivo. In one embodiment, the microbe is contacted with a non-antibiotic agent and an inhibitor of an AcrAB-like efflux pump. In one embodiment, the non-antibiotic agent is selected from the group consisting of: cyclohexadine, quaternary ammonium compounds, pine oil, triclosan, and compound generally regarded as safe (GRAS).
In another aspect, the invention provides a pharmaceutical composition comprising an inhibitor of an AcrAB-like efflux pump and an antibiotic. In one embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. In a preferred embodiment, the antibiotic is selected from the group consisting of fluoroquinolone and rifampin.